In addition to their traditional function of mediating rapid cholinergic transmission, nicotinic acetylcholine receptors (nAChRs) are poised to serve non-traditional functions such as regulating gene transcription through calcium-dependent signaling. Therefore, studying the role that nicotinic signaling plays in guiding the development of the nervous system provides a means for assessing the potential adverse effects of prenatal exposure to drugs such as nicotine. We propose to use the chicken ciliary ganglion as a simple model system in which the role of nicotinic signaling in programmed cell death in vivo can be studied in detail. Programmed cell death normally reduces the total number of neurons in the ciliary ganglion by 50% between embryonic days (E)8 and E14, and embryonic neurons express two functionally distinct populations of nAChRs: homomeric alpha7 containing nAChRs that become extrasynaptically localized by E14, and heteromeric nAChRs containing alpha3, alpha5, Beta2 and Beta4 subunits (alpha3 * nAChRs). Our previous studies suggested that activation of neuronal alpha7 subunit containing nAChRs caused neuronal cell death. We propose that between E6-9, neurons that express a high density of alpha7 nAChRs on their surfaces allow calcium influx that exceeds the set point for survival, thereby inducing apoptosis. After E9, target interactions act to prevent cell death through alpha7 by upregulating an endogenous abtx-like molecule, lynx, that "silences" alpha7 nAChRs. Thus, the central hypothesis of this proposal is that an overabundance of signaling through alpha7 subunit containing nAChRs induces calcium-dependent cell death during normal ciliary ganglion development. To test this hypothesis, the aims of our project are: 1. To determine whether embryonic neurons express more heterogeneity with respect to the levels of alpha7 mRNA prior to cell death and whether neurons expressing elevated alpha7 remain after rescue by MLA and abtx; 2. To determine whether overexpression of alpha7 nAChR subunits causes enhanced levels of intracellular calcium in response to nicotinic stimulation and exacerbates cell death in vivo; 3. To determine whether reducing alpha7 nAChR expression or locally blocking alpha7 nAChRs in ciliary ganglion neurons promotes survival; 4. To test the hypothesis that target- interactions prevent cell death by inducing the expression of lynx, a cell surface molecule with homology to abtx that silences cell surface alpha7 nAChRs. These studies will make a significant contribution towards the detailed understanding of how nicotinic signaling contributes to the development of the nervous system.